| As microbial control agents,entomopathogenic fungi have wide application prospects. Beauveria bassiana,as one of the most common and most important entomopathogenic fungi,has been widely used for fungal insecticide development either in China or outside China.It has wide range host range including many mites and more than 700 insect species belonging to 149 families of 15 orders.It invades insects mainly through cuticle,an incomparable tribute for sucking insects as compared to other insect pathogens.Just as other fungal pesticides,however,the acceptance of B.bassiana products for pest control has been limited,because it kills insects slowly and is usually sensitive to adverse environment and loses their effectiveness rapidly.These drawbacks hinder the utilization of B.bassiana against insects.Currently,genetic engineering techniques are needed to prolong its shelf life and enhance its environmental stability for promotion of application of B.bassiana products.Trehalose exists in a large range of organisms,such as bacteria,fungi,animal and plant.It plays an important role in protection of stress,such as heat,dehydration,drought, freezing,high osmotic pressure,heavy metal salts and toxic substances.There is a strong correlation between accumulation of intracellular trehalose and shelf life of conidia in filamentous fungi.It is speculated that increasing trehalose content in conidia is feasible by genetic engineering techniques to change trehalose metabolism pathway.Heat shock proteins(HSPs) are highly conserved proteins which are synthesized in all organisms responding to environmental stresses.HSP70 is the most important member of them.As a universal cytoprotection protein,HSP70 is ubiquitous molecular chaperone. Moreover,it plays important roles in cellular protection and anti-apoptotic effect.It could help to refold the misfolding proteins and repair the damaged polypeptides.So it is significance to clone hsp70 gene from B.bassiana and study its experssion and action mechanism.The full-length cDNA of two trehalose synthesis relevant genes tps1 and tps2 (GenBank accession No:FJ769373 and FJ769375) were cloned from B.bassiana b202 using SMART RACE RT-PCR.The sequence of their respective upstream promotors (GenBank accession No:FJ769374 and FJ769376) were amplified by genome walking. Sequence analysis showed that there was an intron with size of 55bp in the structural gene of tps1,which was located at the 313th～367th base.The full-length cDNA of tps1 gene was 1906bp,with 1563bp,112bp and 263bp of open reading frame(ORF),5'-untranslated region(5'-UTR) and 3'-untranslated region(3'-UTR),respectively.The open reading frame(ORF) encoded 520 amino acids.The mature protein had a molecular mass of 58.3 kDa with a calculated pI of 5.63.The 3'-UTR contained polyadenylation signal AATATA and 26bp PolyA.The transcription initiation site of B.bassiana tps1 gene was located at G base of position 2785 of upstream sequence.The upstream sequence contained TATA-box, GC-box and several other regulatory elements,such as Oct-1-binding factor, CRE-BP-binding factor,CdxA-binding factor and GATA.There were two introns with size of 140bp and 62bp in the structural gene of tps2,located at the 127th～266th base and 2209th～2270th base,respectively.Their sequences complied with the rule of "5'-GT" and "AG-3'".The full-length cDNA of tps2 gene was 3219bp,with 2619bp,310bp and 290bp of open reading frame(ORF),5'-untranslated region(5'-UTR) and 3'-untranslated region (3'-UTR),respectively.The open reading frame(ORF) encoded 872 amino acids.The mature protein had a molecular mass of 97.8 kDa with a calculated pI of 6.32.The 3'-UTR contained 28bp PolyA.The transcription initiation site of B.bassiana tps2 gene was located at C base of position 4105 of upstream sequence.The upstream sequence contained TATA-box,CAAT-box,GC-box and other regulatory elements,such as cap-binding factor, Adf-1-binding factor,Oct-1-binding factor and GATA.In additional,the content of GATA in the upstream sequence was very high.The same method was used to clone the full-length cDNA of hsp70 gene and its upstream sequence from B.bassiana.Analysis of the complete cDNA cloned,with a whole sequence of 2405bp,showed that it encompassed an open reading frame(ORF) with 1971bp encoding 656 amino acids.The mature protein had a molecular mass of 71.3 kDa with a calculated pI of 4.92.The analyses indicated that the upstream sequence contained several regulatory elements,such as CCAAT-binding factor,GC-box,HSE and GATA,but TATA-box and CAAT-box were not be found.The expression of tps1 and hsp70 gene from B.bassiana under several stress conditions was detected using Realtime-PCR.The effects of different stress conditions on expression of tps1 and hsp70 gene were explored based on mRNA level.The results showed that the change of tps1 gene transcriptional expression in mycelium and conidia of B.bassiana was not significant under three stress conditions,but the expression of hsp70 gene from B.bassiana increased rapidly under the same conditi6ns,suggesting that tpsl and hsp70 gene from B.bassiana have different response mechanisms for external stresses. Tpsl gene into secreting expression vector pPIC9K was cloned and then transformed into Pichia pastoris GS115.After continuous methanol induction,expression protein with molecular weight of ca.60kDa was obtained,which was closed to predicted value and those in related reports.Enzymatic activity assay revealed that the expression protein had bioactivity of trehalose-6-phosphate synthase.The maximum enzymatic activity of crude enzyme liquid was 1.384U/mL. The tpsl gene of B.bassiana was inserted into the cloning site of pBARGPE1 vector, which contained promoter of the glyceraldehyde-3-phosphate gene from Aspergillus nidulans.After the recombinant plasmid was transformed into B.bassiana,tpsl gene was constitutively expressed in the recombinant strain.In comparison with wild-type strains, under lower temperture the shelf life of the recombinant strains was enhanced by nearly 35%.The heat tolerance of recombinant strains was enhanced by nearly 30%within a certain high temperature stress.The UV tolerace of recombinant,however,was not be enhanced.In addition,the growth rate and sporulation of the recombinant strains were lower than those of wild-type ones.It is suggested that constitutive expression of adversity gene of entomogenous fungi enhanced stress resistance of recombinant strains,however, with some negative effects. |
